The relatively random process by which T cell antigen receptors (TCRs) are generated requires that the immune system must have mechanisms to rid itself of T cells that produce self-reactive TCRs. If this process fails the host is faced with autoimmune disease. For self proteins expressed in the thymus, tolerance is achieved primarily by intrathymic physical elimination of self-reactive immature T cells via a programmed cell death mechanism. It is less clear how tolerance is achieved for either tissue- specific self antigens that are not expressed in the thymus or developmentally-regulated proteins that are expressed after the peripheral T cell repertoire is established. Recent work suggests that at least one means by which these types of molecules are tolerated is the induction of functional unresponsiveness, or anergy, in mature peripheral T cells. In vitro experiments indicate that anergy is induced in IL-2- producing T cell clones when certain antigen presenting cells (APCs) present peptide/MHC complexes but do not provide essential costimulatory signals required for IL-2 production. This implies that the type of APC that represents a self antigen, and the environment in which it is presented, could determine whether the responding T cell becomes activated or inactivated. In this proposal, TCR transgenic mice that express on most of their CD4+ T cells a TCR specific for a defined peptide/MHC complex (ovalbumin 323-339/I-Ad) will be used to test in vivo the relationship between peripheral tolerance induction and costimulation by APC. Our preliminary results indicate that a regimen known to induce T cell unresponsiveness in normal mice (peptide injection without adjuvant) also induces T cell unresponsiveness in the TCR transgenic mice. A monoclonal antibody specific for the transgenic TCR will be used to determine if the resulting unresponsiveness is due to deletion of reactive cells, induction of functional anergy, or suppression by regulatory cells. To assess the role of the priming environment, it will be determined whether complete adjuvants antagonize tolerization and whether peptide presentation by costimulation-deficient resting B cells favors tolerization. Cytokine production, delayed-type hypersensitivity, and antibody secretion will be measured to determine whether peripheral tolerance induction affects both Th1 and Th2 cells. Flow cytometric studies will be performed to determine whether unresponsive TCR transgenic T cells express a unique cell surface phenotype and signal transduction studies will be performed to determine whether the unresponsiveness of these cells can be accounted for by proximal TCR signaling defects. The in vivo expression of a novel anergy-associated cytokine will also be tested in unresponsive TCR transgenic mice. The results of these experiments should shed light on the cellular (deletion, anergy, or suppression) and molecular (TCR desensitization, Th1/Th2 cross-regulation) mechanisms underlying peripheral tolerance. An understanding of these mechanisms should allow us to design novel immunosuppressive strategies for specifically inhibiting unwanted immune responses that occur during graft rejection and autoimmune disease.